1. Field of the Invention
The present invention relates to a biochemical sensor chip for simultaneously testing a plurality of biochemical specimens so as to find out whether a substance of interest, such as antibody, antigen, single-stranded DNA, receptor, ligand, enzyme, etc., is present. In particular, the invention relates to a DNA chip and a biochemical sensor chip called a DNA array.
2. Description of Related Art
As the biochemical sensor chip, a DNA chip is known for detecting a plurality of the substances of interest at the same time. This DNA chip is a biochemical sensor chip where a plurality of different DNAs are fixed as probes on a baseplate, such as glass. The probe as mentioned here is a molecule or a substance, which specifically identifies a molecule of interest.
The following methods are known for preparing a DNA chip: A first method by synthesizing DNA probes on a baseplate (Prior Art 1: Science, Vol.251, pp.767-773 (1991)), and a second method by spotting DNA probes already synthesized sequentially (one by one) on a baseplate (Prior Art 2: Science, Vol.270, pp.467-470 (1995)).
According to the first method, on a baseplate made of such as silicon, glass, etc., different types of DNAs of several tens mer are synthesized and founded in one of a plurality of regions by photo-lithography and the technique of photochemical reaction. For instance, a first type of DNA is bonded in a first region, and a second type of DNA is bonded in a second region.
To detect a substance of interest, to-be-tested genes are digested to several tens mer to obtain short fragments. Each fragment is labeled with a fluorescent dye to be added to the surface of the DNA chip. If a DNA fragment (e.g. a cDNA fragment) in complementary relation with the DNA synthesized on the DNA chip is present in the specimen, this DNA fragment is hybridized with the respective DNA on the DNA chip.
After washing off the other DNA fragments, which are not hybridized with the respective DNA on the DNA chip, the DNA chip is examined by an optical detection method with a high sensitivity, such as using a confocal microscopy, and fluorescence signals from the fluorescent dye labeling the DNA fragment in a specific region of the DNA chip are detected. The DNAs bonded in each region of the DNA chip is already known, and the DNA fragment is identified from fluorescence signals detected from each region of the DNA chip.
The second method is a method by spotting DNA probes one by one to each section. In order to facilitate adsorption of DNA, the surface of the baseplate made of such as glass, silicon, polymer material, etc. is coated with a material such as polylysine. Then, a very small quantity of DNA solution is dropped onto the baseplate using a micro-pipette, a syringe, etc. to be dried. As a result, a plurality of spots of DNA probes different from each other are formed on the baseplate. The DNA probes fixed in each of the sections are brought into reaction with to-be-tested genes and modified with the fluorescent substance as described above. Any reacted genes are detected by using any commercially available confocal microscopes or DNA micro-array scanners.
Prior art 3 (JP-A-11-243997) discloses a method by identifying the types of probes attached to the particles based on shape or size of the particle, dielectric property, or color. Light is irradiated on a probe array where the specimen and the particles with reacted probes are arranged two dimensionally. Any signal detected from the passing light through a transparent stage of the probe array by a CCD camera are inputted to a data processing system. After confirming that the particles are not overlapping, the shape of particles (beads) are determined.
Prior art 4 (JP-A-2000-055920) provides a method by arranging polymer microspheres or metal particles already modified with biomolecules, such as DNA, antigen, antibody, receptor, ligand, enzyme, etc. on a baseplate. A baseplate with a gold thin film deposited on the surface is prepared. A template with a plurality of partitions is placed on the baseplate. Then, polystyrene particles suspended in a carbodiimide solution with concentration of 1-50 mM is poured into each of the partitions. Thus, a baseplate is prepared where one layer of different polystyrene particles is adsorbed on each region.
In the DNA chip as described in the prior art, there is problem in uniformity of the capture of probes formed or fixed on the baseplate. Specifically, in each of the methods described in the prior art 1 or 2, the adsorption of the molecules is not always uniform. It is difficult to confirm the adsorbing condition of the molecules by any non-destructive means, and it is almost impossible to determine and evaluate the uniformity of the probes formed or fixed on the baseplate.
The DNA probes on the DNA chip are brought into reaction with the substance of interest, and the substance bonded with DNA probes on the DNA chip is detected. In these processes, no method is known to estimate the number of DNA probes on the baseplate dropped off from the DNA chip. As a result, there is problem in that the intensity of the signal detected from each region of the DNA chip does not accurately reflect the quantity of the substance present in the specimen.
Further, probes are fixed in small regions with a small surface area. As a result, there is problem that the probes cannot be fixed in a quantity necessary to provide a signal with sufficient intensity.
It is an object of the present invention to provide a biochemical sensor for determining number of particles attached (with probes caught on the surface) uniformly fixed in each section and for increasing surface area of the regions to improve sensitivity.
According to the biochemical sensor of the present invention, particles attached with probes for detecting a substance of interest are adsorbed and fixed in each of the sections, which are arranged in a lattice/grid on a baseplate by a chemical patterning method. The particles attached with probes caught in each of the sections are fixed in one single layer and in a very tightly packed state. On the particles fixed in each section, different types of probes are caught. A specimen solution containing the substances of interest labeled with fluorescent substance is poured onto the surface of the baseplate. The substances of interest react and bond to the probes at any sections. By washing the surface of baseplate, un-reacted substances contained in the specimen solution are removed and the substances bonded to the probes attached to the surface of the particles are remained on the baseplate. Different types of the substances of interest labeled with fluorescent substance and bonded to the probes are detected in each section by optically applying a commercially available well-known apparatus for detecting fluorescence emitted from the fluorescent substance excited by irradiation of laser light. Because the particles are fixed in each of sections, apparent surface area per unit area in each section is increased.
Alternatively, one type of substances of interest is served at different densities in each section. There are at least two approaches to attach baseplate sections with different densities of the same kind of substances. One approach is to attach different densities of particles with substantially the same number of probes to the sections. Another approach is to attach the same density of particles each of which has different number of probes to the sections.
The particles attached with probes caught on the surface are prepared in large quantity in advance in a reaction container. Approximately the same number of probes are uniformly caught on each of the particles.
According to the biochemical sensor of the present invention, number of particles fixed in each section on the baseplate is determined by light scattering from the particles. Or, by measuring fluorescent light from fluorescence label of the particles which are labeled in advance, the number of probes in each section can be determined. In each of the processes where the biochemical sensor is used, the quality of the probes in each section of the biochemical sensor is assured, and the substance of interest is detected with high accuracy.
The biochemical sensor of the present invention comprises a plurality of particles attached with approximately the same number of probes each selectively bonding with a substance of interest in a specimen The probes are caught on the surface of the particles, and a planar baseplate has a plurality of sections arranged separately from each other. The particles are fixed so that the number of the particles per unit area in each of the sections is approximately the same, and one layer of the particles is fixed on each of the sections.
The method for manufacturing a biochemical sensor according to the present invention comprises preparing a plurality of particles attached with approximately the same number of probes bonding with the substance of interest in the specimen, having said probes caught on the surface of said particles, and having said particles prepared in different containers for each type of said probes, and having a plurality of section arranged separately from each other on a planar baseplate. The method further comprises fixing a plurality of particles with different types of probes caught on the surface of the particles such that the number of the particles per unit area in each of the sections is approximately the same, or fixing one layer of particles where different types of probes are caught. The method is also characterized in that the number of the particles fixed per unit area in each of the sections is non-destructively determined by irradiating light to regions in each of the sections where the particles are fixed.
Different substances of the same type, such as DNAs, RNAs, proteins, etc., are extracted from different sources, such as different patients. For detecting different types of substances, such as DNAs, RNAs, proteins, etc., different baseplates are used, i.e., one baseplate for detecting one type of substances. For example, if a DNA is used as a probe, all of the probes fixed on a baseplate through particles are DNAs. It is not suggested to mix probes of different types on a single baseplate.
There is no description regarding the above features in any of the prior art references. A typical example of the probe in the present invention is a single-stranded DNA, and typical examples of the particles include polymer particles (such as polystyrene particles) and glass particles.
In the above description, the expression that xe2x80x9cthe particles attached with substantially the same number of probes on the surfacexe2x80x9d are defined as follows. It is assumed that the number of probes caught on the surface of the particles (i=1, 2, . . . , N) is Xi=X1, X2, . . . , XN respectively, and that average value of Xi=X1, X2, . . . , XN is Xav. Then, at statistical probability of 95%, number of probes 50% above or below the average value Xav (i.e. number of probes within the range of 0.5 Xavxe2x88x921.5 Xav) are caught on the particles. Specifically, if number of probes Xi caught on the particles xe2x80x9cixe2x80x9d is within the range of 0.5 Xavxe2x88x921.5 Xav, the particles xe2x80x9cixe2x80x9d can be regarded as the particles attached with approximately the same number of probes on the surface.
Also, in the above description, the expression that xe2x80x9cnumber of particles per unit area in each of the sections, i.e. density, is substantially the samexe2x80x9d is defined as follows: It is assumed that the number of particles per unit area fixed in sections (i=1, 2, . . . , M) is Yi=Y1, Y2, . . . , YM respectively, and that average value of Yi=Y1, Y2, . . . , YM is Yav. Then, at statistical probability of 95%, it is defined as where the number of the particles falls 50% above or below the average value Yav (i.e. number of the particles in the range of 0.5 Yavxe2x88x921.5 Yav) as xe2x80x9csubstantially the samexe2x80x9d. That is, if the number Yi of the particles fixed in the section xe2x80x9cixe2x80x9d is within the range of 0.5 Yavxe2x88x921.5 Yav, the section xe2x80x9cixe2x80x9d can be regarded as a section with substantially the same number of particles fixed.
Other and further objects, features and advantages of the invention will appear more fully from the following description.